Protecting the Achilles heel: three FolE_I-type GTP-cyclohydrolases needed for full growth of metal-resistant Cupriavidus metallidurans under a variety of conditions

ABSTRACT In Cupriavidus metallidurans and other bacteria, biosynthesis of the essential biochemical cofactor tetrahydrofolate (THF) initiates from guanosine triphosphate (GTP). This step is catalyzed by FolE_I-type GTP cyclohydrolases, which are either zinc-dependent FolE_IA-type or metal-promiscuous FolE_IB-type enzymes. As THF is also essential for GTP biosynthesis, GTP and THF synthesis form a cooperative cycle, which may be influenced by the cellular homeostasis of zinc and other metal cations. Metal-resistant C. metallidurans harbors one FolE_IA-type and two FolE_IB-type enzymes. All three proteins were produced in Escherichia coli. FolE_IA was indeed zinc dependent and the two FolE_IB enzymes metal-promiscuous GTP cyclohydrolases in vitro, the latter, for example, functioning with iron, manganese, or cobalt. Single and double mutants of C. metallidurans with deletions in the folE_I genes were constructed to analyze the contribution of the individual FolE_I-type enzymes under various conditions. FolE_IA was required in the presence of cadmium, hydrogen peroxide, metal chelators, and under general metal starvation conditions. FolE_IB1 was important when zinc uptake was impaired in cells without the zinc importer ZupT (ZIP family) and in the presence of trimethoprim, an inhibitor of THF biosynthesis. FolE_IB2 was needed under conditions of low zinc and cobalt but high magnesium availability. Together, these data demonstrate that C. metallidurans requires all three enzymes to allow efficient growth under a variety of conditions. IMPORTANCE Tetrahydrofolate (THF) is an important cofactor in microbial biochemistry. This “Achilles heel” of metabolism has been exploited by anti-metabolites and antibiotics such as sulfonamide and trimethoprim. Since THF is essential for the synthesis of guanosine triphosphate (GTP) and THF biosynthesis starts from GTP, synthesis of both compounds forms a cooperative cycle. The first step of THF synthesis by GTP cyclohydrolases (FolEs) is metal dependent and catalyzed by zinc- or metal-promiscuous enzymes, so that the cooperative THF and GTP synthesis cycle may be influenced by the homeostasis of several metal cations, especially that of zinc. The metal-resistant bacterium C. metallidurans needs three FolEs to grow in environments with both high and low zinc and cadmium content. Consequently, bacterial metal homeostasis is required to guarantee THF biosynthesis.

The strains were cultivated in medium zinc TMM (M1) with or without 100 µM ethylene diamine tetraacetate (EDTA), 10 µM dipyridyl (DIP, also bipyridyl) or 1 µM N,N,N′,N′-tetrakis(2pyridinylmethyl)-1,2-ethanediamine (TPEN) and the specific beta-galactosidase activity was determined.Bold-faced, if different (D>1) in the comparisons (i) AE104 parent with additions to no additions or (ii) mutants to parent AE104 under the same conditions; n.d.not done; n ≥ 3, deviations shown.S4.Metal content of C. metallidurans strain AE104 and mutants with deletions in the genes for FolE_I-type enzymes after cultivation under iron and zinc starvation conditions a .

Supplementary Table
a The metal content in metals per cell was determined with the ICP-MS of C. metallidurans strain AE104 and its ∆folE_IA, ∆folE_IB1 and ∆folE_IB2 mutant in medium zinc TMM (M1, mZn) or low iron TMM (M3, lFe, no SL6 and no iron)).The values for M1 are the same as in Table 5 and listed here again to facilitate the comparison.Bold, difference to the AE104 value in M1 with D>1.Three biological repeats, deviations indicated, n.a., not analyzed.a The metal content in metals per cell was determined with the ICP-MS of C. metallidurans strain AE104, ∆zupT and its ∆folE_IA, ∆folE_IB1 and ∆folE_IB2 mutant in medium zinc TMM (M1) in the presence of either 0.1 mM EDTA or 25 µM DIP.The metal content without additions is given for reference.Bold-faced numbers are different (D > 1) from the AE104 value; n.d., no data since below the detection limit.Three biological repeats, deviations indicated.Exceptions: b no differences and therefore not repeated; c one result, repeats below detection limit, d two results, one repeat below detection limit, therefore not bold-faced despite the difference to the AE104 value.a The metal content in metals per cell was determined with the ICP-MS of C. metallidurans strain AE104, ∆zupT and its ∆folE_IA, ∆folE_IB1 and ∆folE_IB2 mutant in medium zinc TMM (M1) in the presence of 1 µM Cd(II).The metal content without additions is given for reference.Bold-faced numbers are different (D > 1) from the value of the respective parent; n.d., no data since below the detection limit.Three biological repeats, deviations indicated.Exceptions: b one result, repeats below detection limit, c two results, one repeat below detection limit, therefore not bold-faced despite the difference to the AE104 value.

Supplementary Table S7. Growth rates (h
Supplementary Table S12

Rmet_2917 panB
Op0825f_3 Q1LJ86 3-methyl-2-oxobutanoate hydroxymethyltransferase a The data were compiled from data base submissions connected to several publications (1-3) and EcoCyc (4).b Locus tag bold: uses THF; c gene name bold, gene products were identified, quantified, and more than 100 copies per cell were found (2).d (3).e Q, Q(AE104: EDTA/Zn), regulation of the respective gene in EDTA-treated C. metallidurans AE104 cells compared to those cultivated in the presence of zinc (1), bold up-and bold-italics down-regulated.For the operons, under RpoD control bold, not under RpoD control on a grey field, both (two promoters) bold and on a grey field, weak RpoD motif normal, no transcriptional start site identified in italics (5).S13

Supplementary Table S2. Activity folE_IA and folE_IB1 fusions in the presence or absence inhibitors of the THF biosynthesis a .
a The strains were cultivated in medium zinc TMM (M1) with or without additives at the indicated concentrations (YE, yeast extract).Bold faced number: different (D > 1) in the comparisons (i) AE104 with/without additions; (ii) ∆zupT, ∆zur compared to AE104; (iii) other mutants compared to their respective parent under the same condition.With the exception of three experiments, n ≥ 3 biological repeats, mean values and deviations indicated; n.d., not done.bTheresult of one biological replicate only, mean values and deviations from the technical repeats.Not further pursued because of the lack of any difference c The results of two biological replicates, mean values and deviations from the technical and biological repeats.Also not pursued because of the lack of any difference compared to the control.aThestrainswere cultivated in medium zinc TMM (M1) with or without sulfonamide (SUAM) or trimethoprim (TMP) at the indicated concentrations.Both inhibit later steps of the tetrahydrofolate (THF) biosynthesis, the dihydropteroate synthase and dihydrofolate reductase, respectively; n ≥ 3, deviations shown Supplementary TableS3.

-1 ) in the presence of 100 µM EDTA Supplementary Table S8. IC50 values for TPEN and DIP
Bold faced number: different (D > 1) from the respective AE104 value.

Table S10 . IC50 values for cadmium, cobalt, paraquat (PQ) and hydrogen peroxide
Bold faced number: different (D > 1) from the respective value of the parent AE104 or ∆zupT.